Hormone-supplemented defined media able to support the growth and differentiation of several embryonal carcinoma (EC) cell lines are being developed in order to identify and study the extra-cellular signals that induce differentiation. Previous studies have demonstrated that the "nullipotent" EC cell line F9, can be grown in a defined medium (EM-3) containing fibronectin, insulin and transferrin. The F9 cells do not differentiate in EM-3 unless exposed to an inducer such as retinoic acid. Current studies indicate that multipotent EC cells do not proliferate in EM-3. In direct contrast to F9, 3 different multipotent EC cell lines rapidly differentiate when cultured in EM-3 and the differentiated cells that form continue to proliferate for several generations provided EM-3 is supplemented with high density lipoprotein. In each case the entire EC cell population differentiates in the defined medium and one major cell type forms - greater than 85% of the cells exhibit the properties of parietal extraembryonic endoderm. Attempts to understand these results led to the finding that fibronectin and laminin induce multipotent EC cells to differentiate and provide the first evidence that these molecules may directly influence the appearance of extraembryonic endoderm during early mammalian development.